Polycomb-group (PcG) proteins comprise an epigenetic system that maintains the transcriptional repression of target genes. Originally identified as negative regulators of Drosophila Hox genes, their functions and molecular/biochemical activities have been highly conserved from insects to mammals. Mammalian PcG orthologs are required to maintain the pluripotent state of stem cells and their misexpression contributes to oncogenesis. PcG proteins do not initiate transcriptional repression, but rather take over repression from gene-specific transcription factors. Once PcG- mediated repression is established, it is then capable of stably and heritably maintaining gene silence through numerous cell cycles. During the maintenance phase of PcG-mediated repression, DNA binding PcG proteins (e.g., Pleiohomeotic) bind to sequences within Polycomb Response Elements (PREs) and recruit additional PcG protein complexes, such as PRC2, which in turn methylates histone H3 at lysine 27 (H3K27me3), facilitating recruitment of PRC1. Virtually nothing is known about the mechanisms by which PcG proteins initially recognize the repressed state of target genes and the molecular steps that lead to establishment of PcG-mediated repression. In large part, this is due to the heterogeneous expression of target genes within embryos during the developmental stages when PcG-mediated repression is established. In the proposed studies, genetic manipulation will be used to obtain syncytial and cellular blastoderm stage embryos in which a PcG target gene is ubiquitously repressed at the syncytial blastoderm stage by a maternally expressed transcription factor, and in which the PcG is required to maintain its ubiquitous repression by the cellular blastoderm stage. Two specific aims are proposed: (1) Map the locations of PREs within the regulatory region of the PcG target gene;(2) Define the physical and temporal distributions of regulatory proteins, including PcG proteins, at the PcG target gene during the developmental window when PcG-mediated repression is established. The goal of these studies is to provide insight into the molecular and biochemical steps involved in the transition from initial transcription factor-mediated repression to PcG-mediated maintenance of transcriptional silence. These results will be particularly relevant to the generation of iPS cells and oncogenesis, both of which involve de novo establishment of PcG-mediated repression. PUBLIC HEALTH RELEVANCE: Polycomb-group proteins are highly conserved negative regulators of gene expression, which maintain the silence of numerous developmentally important genes in organisms ranging from fruit flies to humans. Overexpression of Polycomb-group proteins contributes to a range of human cancers, including metastatic prostate and aggressive breast cancers as well as hematologic and epithelial cancers. The proposed studies will provide greater insight into the molecular mechanisms by which Polycomb-group- mediated repression is initially established, which may lead to the development of therapies that inhibit their activities, blocking their oncogenic effects.